The advent of ultrashort laser light pulses as a laboratory tool provides an opportunity to probe and manipulate anatomy and function in nervous systems. Ultrashort pulses are the essential means to drive the nonlinear absorption of light by biomolecules, which leads to a localized region of excitation and forms the basis of two- photon scanning microscopy. More recently, nonlinear absorption has been exploited as a means to reliably and reproducibly create micrometer-sized ablations in brain tissue with minimal collateral damage. These ablations drive all-optical histology, which allows anatomy to be imaged with micrometer resolution and mapped throughout an entire brain. They can also be used to perturb neocortical blood flow as a means to probe normal and diseased vascular function. Yet much additional effort is required to advance this new technology as a means to enable studies of neuronal and vascular architectonics. Our technical program is focused on advancing the mixture of ablation and imaging. [unreadable] Optimization of pulsed lasers for nonlinear ablation. [unreadable] Optimization of objectives for high efficiency, in vivo ablation and imaging. [unreadable] Design and optimization of algorithms to reconstruct and analyze vascular and large-scale cellular anatomy. Our scientific effort involves the study of structural and functional anatomy in the brain. [unreadable] Automatic mapping of the relation among neuronal and nonneuronal nuclei, vasculature, and mitochondria or other subcellular structures within the rodent brain. This includes stroke-induced changes in angioarchitecture. [unreadable] Optical induction of localized,thrombotic and hemorrhagic strokes as a means to study the redistribution of blood flow and blood 02 in response to perturbations in brain homeostasis. The program will be carried by a partnership of three research groups with a history of collaboration: Kleinfeld with expertise in nonlinear imaging and cellular/systems neuroscience; Squier with expertise in laser and microscope design and biological imaging; and Ifarraguerri with expertise in algorithm design and biomedical imaging. The proposed advancement in nonlinear optical methods will yield novel tools for manipulating and probing tissues. We will make these tools reliable and readily available to the biomedical community. The proposed model systems may lead to improvements in preclinical models to assay therapeutics for stroke. 3ERFORMANCE SITE(S) (organization, city, state) Department of Physics Department of Physics Science Applications International Corp. University of California at San Diego Colorado School of Mines 4001 North Fairfax Drive La Jolla, CA 92093-0319 Golden, CO 80401 Arlington, VA 22203 KEY PERSONNEL. See instructions on Page 11. Use continuationpages as neededto provide the required information in the format shown below. Name Organization Role on Project Kleinfeld, David Physics Dept., UCSD Principal Investigator Ifarraguerri, Augustin Science Applications International Corp. Co-Investigator Squier, Jeff Physics Dept. Colorado School of Mines Co-Investigator Dolnick, Earl Friedman, Beth O'Brien, Gary Schaffer, Chris B. Thompson, Beverly Tsai, Philbert Physics Dept., UCSD Engineer Neurosciences Dept., UCSD Project Scientist Science Applications International, Inc. Programmer Physics Dept., UCSD Project Scientist Science Applications International Corp. Senior Scientist Physics Dept., Colorado School of MinesPostdoctoral Researcher Principal Investigator/ProgramDirector (Last, first, middle): _Kleinfeld, David Type the name of the principal investigator/program director at the top of each printed page and each continuation page. (For type specifications, see instructions on page6.) RESEARCH GRANT TABLE OFCONTENTS Page Numbers Face Page 1 Description,